The activation of prothrombin to thrombin in a purified system is a multi-step process comprising two Factor Xa-catalyzed reactions which propagate thrombin and several thrombin-catalyzed feedback loops. In this study, independent kinetic constants for each step will be derived, to determine which reactions predominate in plasma by virtue of their KM and kcat values. Various methods will be used to make initial rate measurements, each based on the appearance of a unique product at a low concentration of protease. The cleavage of prothrombin by thrombin will be estimated from the release of radiolabeled fragment 1. Cleavage of prothrombin by Factor Xa will be determined from the generation of fragment 1.2 in the presence of a thrombin inhibitor. The kinetics of thrombin formation from its immediate precursor prethrombin 2 and from whole prothrombin will be obtained with a discontinuous amidolytic assay. Many criteria govern the rate of thrombin formation. They include the presence of cofactors (Factor V, phospholipids and Ca2 ion) and activation fragments, and the structural integrity of the participating proteins. The effects of these on the kinetic constants will be investigated to show how they take part in normal catalysis and binding. These data are significant for several reasons. First, they are of inherent interest because few kinetic constants for proteolysis are known. Second, prothrombin activation may be a model for other proteolyses requiring protein cofactors. Third, the control of thrombin yield is crucial in hemostasis: thrombin action leads not only to fibrin formation and cross-linking, but also causes the platelet release reaction and, by its action on Factor VIII governs the extent of coagulation by the intrinsic pathway.